scholarly journals Validation of Nonnested and Real-Time PCR for Diagnosis of Sheep-Associated Malignant Catarrhal Fever in Clinical Samples

2007 ◽  
Vol 19 (4) ◽  
pp. 405-408 ◽  
Author(s):  
Donald L. Traul ◽  
Naomi S. Taus ◽  
J. Lindsay Oaks ◽  
Donal O' Toole ◽  
Fred R. Rurangirwa ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
High Specificity ◽  
Clinical Samples ◽  
Malignant Catarrhal Fever ◽  
Fatal Disease ◽  
Tissue Samples ◽  
Specificity And Sensitivity ◽  
Experimentally Induced

Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.

scholarly journals Comparison of real-time PCR protocols in detection and quantification of fruit tree 16SrX group phytoplasmas

Genetika ◽  
2016 ◽  
Vol 48 (2) ◽  
pp. 629-642 ◽  
Author(s):  
Tomas Kiss ◽  
Tomas Necas ◽  
Jana Necasova
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Fruit Tree ◽  
Amplification Efficiency ◽  
Specificity And Sensitivity ◽  
Intercalating Dye ◽  
Detection Characteristics ◽  
Detection And Quantification

In this work, two real-time PCR protocols based on intercalating dye and two on hydrolysis probes were tested using field collected fruit tree samples infected by 16SrX group (AP, PD and ESFY) phytoplasmas. Specificity and sensitivity of protocols and amplification efficiency were the main testing parameters. Results of real-time PCR protocols were compared to nested PCR. All real-time PCR protocols confirmed their specificity of detection. All real-time PCR protocols were 10-100 times more sensitive than nested PCR. Afterall real-time PCR protocols based on hydrolysis probes were 10 times more sensitive than protocols based on intercalating dyes. Among protocols based on hydrolysis probes, slightly better detection characteristics were shown by protocol by CHRISTENSEN et al. (2004).

scholarly journals Detection of a new Non-Classified Chlamydia Species in Hens in Poland

2013 ◽  
Vol 57 (1) ◽  
pp. 25-28 ◽  
Author(s):  
Monika Szymańska-Czerwińska ◽  
Krzysztof Niemczuk ◽  
Konrad Sachse ◽  
Agata Mitura ◽  
Teresa Agnieszka Karpińska ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Subcutaneous Tissue ◽  
Taxonomic Status ◽  
Tissue Samples ◽  
Intracytoplasmic Inclusions ◽  
Electron Microscopy Analysis ◽  
Provinces Of Poland ◽  
Microscopy Analysis

Abstract The outbreak of chlamydiosis in one of the western provinces of Poland, was diagnosed accidentally as a concurrent infection in a commercial laying hen flock during an outbreak of fowl pox. For histological examination, skin and subcutaneous tissue samples from lesions on heads of the birds were collected. Swabs from throat and trachea have been examined by nested PCR, real-time PCR, and partial ompA sequencing. Detailed electron microscopy analysis revealed fowl pox intracytoplasmic inclusions, called Bollinger bodies, and the presence of other intracytoplasmic inclusions; specific for Chlamydia sp. Results of nested PCR confirmed the presence of Chlamydiaceae sp. in two tested samples. Surprisingly, one of the two Chlamydiaceae-positive cases turned out to be infected with a non-classified strain. Results of real-time PCR and sequencing confirmed the presence of a new Chlamydia species that has not been found in Poland to date. Partial sequencing and BLAST analysis of ompA gene sequence confirmed the highest homology to non-classified poultry strains of Chlamydia sp. that were previously detected in Germany and France. The zoonotic potential and the exact taxonomic status of this atypical strain have yet to be defined.

Comparison of the Specificity and Sensitivity of PCR, Nested PCR, and Real-Time PCR for the Diagnosis of Histomoniasis

2005 ◽  
Vol 49 (3) ◽  
pp. 366-370 ◽  
Author(s):  
H. M. Hafez ◽  
R. Hauck ◽  
D. Lüschow ◽  
L. McDougald
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Specificity And Sensitivity

scholarly journals The Application of a Nanomaterial Optical Fiber Biosensor Assay for Identification of Brucella Nomenspecies

Biosensors ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 64 ◽  
Author(s):  
Kelly McCutcheon ◽  
Aloka B. Bandara ◽  
Ziwei Zuo ◽  
James R. Heflin ◽  
Thomas J. Inzana
Keyword(s):  
Optical Fiber ◽  
Light Transmission ◽  
High Specificity ◽  
Clinical Samples ◽  
Long Period Grating ◽  
Tissue Samples ◽  
Spleen Tissue ◽  
Tissue Extracts ◽  
Specificity And Sensitivity ◽  
Detection Assay

Bacteria in the genus Brucella are the cause of brucellosis in humans and many domestic and wild animals. A rapid and culture-free detection assay to detect Brucella in clinical samples would be highly valuable. Nanomaterial optical fiber biosensors (NOFS) are capable of recognizing DNA hybridization events or other analyte interactions with high specificity and sensitivity. Therefore, a NOFS assay was developed to detect Brucella DNA from cultures and in tissue samples from infected mice. An ionic self-assembled multilayer (ISAM) film was coupled to a long-period grating optical fiber, and a nucleotide probe complementary to the Brucella IS711 region and modified with biotin was bound to the ISAM by covalent conjugation. When the ISAM/probe duplex was exposed to lysate containing ≥100 killed cells of Brucella, or liver or spleen tissue extracts from Brucella-infected mice, substantial attenuation of light transmission occurred, whereas exposure of the complexed fiber to non-Brucella gram-negative bacteria or control tissue samples resulted in negligible attenuation of light transmission. Oligonucleotide probes specific for B. abortus, B. melitensis, and B. suis could also be used to detect and differentiate these three nomenspecies. In summary, the NOFS biosensor assay detected three nomenspecies of Brucella without the use of polymerase chain reaction within 30 min and could specifically detect low numbers of this bacterium in clinical samples.

scholarly journals Multiplex real-time PCR assay combined with rolling circle amplification (MPRP) using universal primers for non-invasive detection of tumor-related mutations

RSC Advances ◽  
2018 ◽  
Vol 8 (48) ◽  
pp. 27375-27381 ◽  
Author(s):  
Jian Gong ◽  
Yishuai Li ◽  
Ting Lin ◽  
Xiaoyan Feng ◽  
Li Chu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rolling Circle Amplification ◽  
High Specificity ◽  
Universal Primers ◽  
Multiplex Detection ◽  
Pcr Assay ◽  
Rolling Circle ◽  
Non Invasive ◽  
Specificity And Sensitivity

The MPRP system for SNP discrimination was developed, which showed high specificity and sensitivity for multiplex detection of tumor-related mutations.

Real-Time PCR Method for Quantification of Staphylococcus aureus in Milk

2007 ◽  
Vol 70 (1) ◽  
pp. 90-96 ◽  
Author(s):  
M. GOTO ◽  
H. TAKAHASHI ◽  
Y. SEGAWA ◽  
H. HAYASHIDANI ◽  
K. TAKATORI ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Real Time ◽  
Real Time Pcr ◽  
Pure Culture ◽  
High Specificity ◽  
Pcr Assay ◽  
Milk Samples ◽  
High Temperature Short Time ◽  
Specificity And Sensitivity ◽  
Pcr Method

A reproducible real-time PCR method that targets the putative transcriptional regulator gene of Staphylococcus aureus was developed to quantify this microorganism in milk samples. On the basis of partial sequences of this gene determined from S. aureus strains, we designed the specific primers and probe for use in a quantitative PCR assay. These specificities were confirmed with 25 strains of S. aureus and 35 strains of other bacteria. A real-time PCR assay with serial 10-fold dilutions of purified DNA and pure culture was conducted. It was possible to construct standard curves with a high correlation coefficient (r2 = 0.99) in the range of 50 ng to 50 fg for purified DNA and 107 to 101 CFU/ml for a pure culture. The constructed standard curve for milk samples was similar to that for the pure culture, and the quantification of S. aureus in the range of 107 to 101 CFU/ml was possible. Moreover, to determine how our real-time PCR method would perform under actual analytical conditions, we quantified the DNA from S. aureus after two types of heat treatments were used for the pasteurization of milk. The amount of DNA found was affected after heat treatment at 63°C for 30 min (low-temperature long-time method) but not at 72°C for 15 s (high-temperature short-time method). The results indicate that the real-time PCR method developed in this study is effective for monitoring S. aureus contamination in milk because of its high specificity and sensitivity.

scholarly journals Establishment of a Duplex Real-time qPCR Method for Detection of Salmonella Spp. And Serratia Fonticola in Fishmealestablishment of a Duplex Real-time qPCR Method for Detection of Salmonella Spp. And Serratia Fonticola in Fishmeal

2020 ◽  
Author(s):  
Jinghua Ruan ◽  
Wujun Wang ◽  
Tiying Zhang ◽  
Teng Zheng ◽  
Jing Zheng ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
High Specificity ◽  
Clinical Samples ◽  
Amplification Efficiency ◽  
Salmonella Spp ◽  
Specificity And Sensitivity ◽  
Qpcr Method ◽  
Real Time Qpcr

Abstract Salmonella spp. is a high-risk bacterial pathogen that is monitored in imported animal-derived feedstuffs. Serratiafonticola is the bacterial species most frequently confused with Salmonella spp. in traditional identification methods based on biochemical characteristics, which are time-consuming and labor-intensive, and thus unsuitable for daily inspection and quarantine work. In this study, we established a duplex real-time qPCR method with invA- and gyrB-specific primers and probes corresponding to Salmonella spp. and S. fonticola. The method could simultaneously detect both pathogens in imported feedstuffs, with a minimum limit of detection for Salmonella spp. and S. fonticola of 197 copies/SL and 145 copies/SL, respectively (correlation coefficient R2 = 0.999 in both cases). The amplification efficiency for Salmonella spp. and S. fonticola was 98.346% and 96.49%, respectively. Detection of clinical samples was consistent with method GB/T 13091-2018, and all seven artificially contaminated imported feed samples were positively identified. Thus, the developed duplex real-time qPCR assay displays high specificity and sensitivity, and can be used for the rapid and accurate detection of genomic DNA from Salmonella spp. and S. fonticola within hours. This represents a significant improvement in the efficiency of detection of both pathogens in imported feedstuffs.

scholarly journals Development of a real-time PCR assay for the detection of Brucella ovis DNA in clinical samples

2021 ◽  
Vol 7 (1-2) ◽  
pp. 17-20
Author(s):  
N. V. Marchenko ◽  
O. Yu. Lymanska ◽  
A. P. Gerilovych ◽  
V. I. Bolotin
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Closely Related Species ◽  
Tissue Samples ◽  
Fast Detection ◽  
Brucella Ovis ◽  
Direct Indication ◽  
Pcr Targeting

The etiological agent of infectious ovine epididymitis is Brucella ovis and for its direct indication in clinical samples several PCR protocols are proposed. This study describes a design and selection of the oligonucleotides for real-time PCR targeting conservative BOV_A0504 gene. The specificity of a real-time PCR was validated using 25 B. ovis field isolates and 14 microorganisms of closely related species. The detection limit of B. ovis in bacterial culture was determined as 3.5×101 CFU/mL with Ct value of 37.8. There are no detectable fluorescence signals in the clinical samples from intact animals, whereas bacteriologically confirmed material such as urine and testicle tissue samples were positive. It confirms that the assay is highly specific for detection of B. ovis DNA. Thus, the proposed real-time PCR assay enables fast detection and quantification of B. ovis in clinical material, which can be used as additional test for estimation of the health status of a sheep herd

Fast Real-Time PCR assay for detection of Tetramicra brevifilum in cultured turbot

Parasitology ◽  
2012 ◽  
Vol 140 (3) ◽  
pp. 338-342 ◽  
Author(s):  
MERCEDES ALONSO ◽  
FÁTIMA C. LAGO ◽  
MARÍA GÓMEZ-REINO ◽  
JACOBO FERNÁNDEZ ◽  
IRIS MARTÍN ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Infection Intensity ◽  
High Specificity ◽  
Life Cycle Stage ◽  
Microsporidian Parasite ◽  
Detection And Identification ◽  
Specificity And Sensitivity ◽  
Spore Longevity ◽  
Severe Infections

SUMMARYGlobal aquaculture production of turbot has rapidly increased worldwide in the last decade and it is expected to have even bigger growth in the next years due to new farms operating. The losses caused by pathogen infections have grown at the same time as the production of this species. Parasitological infections are among the main relevant pathologies associated with its culture and produce serious losses in aquaculture, reduce the growth rate in fish and may lead to unmarketable fish due to skeletal muscle abnormalities in cases with high intensity of infection. The microsporidian parasite Tetramicra brevifilum causes severe infections and generates major losses in farmed turbot. Infections are difficult to control due to spore longevity and its direct transmission. To facilitate the infection management, an effective tool for fast detection and identification of T. brevifilum is needed. This study provides a molecular methodology of fast Real-Time PCR for T. brevifilum detection to the aquaculture industry, useful for routine control of T. brevifilum at turbot farms. The method is characterized by its high specificity and sensitivity, and it can be applied to cultured turbot for parasite detection regardless of the life-cycle stage of the pathogen or the infection intensity.

scholarly journals Comparative aspects of laboratory testing for the detection of Toxoplasma gondii and its differentiation from Neospora caninum as the etiologic agent of ovine abortion

2020 ◽  
Vol 32 (6) ◽  
pp. 898-907
Author(s):  
Nicola Meixner ◽  
Marie F. Sommer ◽  
Nelly Scuda ◽  
Kaspar Matiasek ◽  
Matthias Müller
Keyword(s):  
Toxoplasma Gondii ◽  
Real Time ◽  
Real Time Pcr ◽  
Neospora Caninum ◽  
Placental Tissue ◽  
Etiologic Agent ◽  
Detection Methods ◽  
Tissue Samples ◽  
Specificity And Sensitivity ◽  
Histologic Evaluation

Histologic examination of aborted material is an essential component in the diagnosis of ovine toxoplasmosis. However, the detection of Toxoplasma gondii in histologic sections, and its differentiation from the closely related protozoan Neospora caninum, is challenging. We developed a chromogenic in situ hybridization (ISH) assay for the identification of T. gondii in paraffin-embedded tissue samples. We examined retrospectively the archived placental tissue of 200 sheep abortion submissions for the presence of T. gondii by immunohistochemistry (IHC), ISH, and real-time PCR (rtPCR). All placental samples that tested positive for T. gondii by rtPCR (9 of 200) were also positive by IHC, with inconclusive IHC staining in an additional 7 rtPCR-negative cases. Further testing for N. caninum of all 200 placentas by rtPCR revealed 7 Neospora-positive cases. T. gondii ISH was positive in 4 of 9 IHC-positive samples and 1 of the 7 N. caninum rtPCR-positive samples. Real-time PCR was used as the reference standard for specificity and sensitivity calculations regarding placenta samples. Specificity of ISH and IHC was 99% and 96–100%, respectively. The sensitivity of ISH (44%) was quite low compared to IHC (100%). The exclusive use of ISH for the detection of T. gondii, and thus for the diagnosis of ovine toxoplasmosis, was not acceptable. However, combined with rtPCR, both ISH and IHC can be useful detection methods to improve histologic evaluation by visualizing the parasite within tissue sections.

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